Meningococcus: Introduction, Culture characteristics, Pathogenesis,

Meningococcus: Introduction, Culture characteristics, Pathogenesis, Laboratory Diagnosis and Treatment

Introduction of Meningococcus

Meningococcus is the common name of Neisseria meningitidis and causes meningitis (infections of the lining of the brain and spinal cord) and bloodstream infections (bacteremia or septicemia). The genus Neisseria is named after the German bacteriologist Albert Neisser, who discovered Neisseria gonorrhoeae, the pathogen that causes the human disease gonorrhea in 1879. Later, in 1887 Weichselbaun isolated meningococcus from the cerebrospinal fluid (CSF) of a patient. Meningococcus spreads through the exchange of respiratory and throat secretions like spit (e.g. by living in close quarters, kissing). Antimicrobial agents, as well as vaccine both, are available to save a life from this bacterial infections.

Scientific classification of Meningococcus

Domain: Bacteria

Phylum: Proteobacteria

Class: Betaproteobacteria

Order: Neisseriales

Family: Neisseriaceae

Genus: Neisseria

Species: Neisseria meningitidis

The Neisseria are Gram negative diplococci.

N. meningitidis and N. gonorrhoeae are the pathogenic species of this genus.

General characteristics of Neisseria meningitidis

Following are the general features of N. meningitidis-

They are Gram-negative spherical, oval or cocci, bean-shaped (adjacent sides flattened), in pairs or diplococci.
Do not possess flagella or spores
They are capsulated and possess pili.
Strict parasites, do not survive long outside of the host
They are strict aerobes and thus can not grow anaerobically.
Oxidative metabolism
Produce enzymes catalase and oxidase
Pathogenic species require enriched complex media and CO₂.

Morphology of Neisseria meningitidis

They are Gram-negative spherical, oval, or cocci, 0.8 x 0.6 µm in size arranged in pairs with the adjacent sides flattened. sides flattened).
They do not possess flagella or spores.
They are capsulated and possess pili.

Cultural characteristics of Neisseria meningitidis

They do not grow on ordinary media like nutrient agar but have exacting growth requirements but can grow in non-selective media like blood agar, Chocolate agar, and Mueller-Hinton starch casein hydrolysate agar. Growth is improved by the addition of blood or serum. Growth is also improved by incubation in the presence of 5- 10 % CO₂. Growth temperature is 35-36⁰C and pH ranges of 7.4-7.6. Colonies are 1-2 mm in diameter, convex, grey, and transparent. No hemolysis in blood agar. Selective media Modified Thayer-Martin medium with antibiotics ( vancomycin, colistin, nystatin, and trimethoprim) and New York City medium. Colony characteristics on Modified Thayer-Martin medium are as follows-

Color: Bluish grey
Shape: Round
Size: About 1 mm
Surface: Smooth
Elevation: Convex
Opacity: Translucent
Consistency: Butyrous

Biochemical properties of N. meningitidis

They are oxidase-positive; i.e., they possess the enzyme cytochrome and produce oxidase. Meningococcus is a maltose fermenter and does not produces beta-lactamases. It has three important virulence factors: 1. Polysaccharide capsule. It is antiphagocytic in nature. 2. The endotoxin of N. meningitidis is a lipopolysaccharide (LPS). It induces septic shock by causing the release of cytokines. 3. IgA protease. It cleaves the IgA antibodies present in the respiratory mucosa.

Pathogenesis of Meningococcus

• Humans are the only natural hosts and they are transmitted by airborne droplets, They colonize the nasopharynx and become transient flora of the upper respiratory tract. From the nasopharynx, the organism can enter the bloodstream and spread to the meninges, and grow in the cerebrospinal fluid (CSF). N. Meningitidis is the most common cause of meningitis in persons between the ages of 2 and 18 years. Outbreaks of meningitis are most common in winter and early spring and favored by close contact between individuals. They cause meningitis and meningococcemia (multiplication of bacteria in the bloodstream).

Clinical Features

Febrile illness: Mild and self-limiting

Pyogenic meningitis: High fever, stiff neck, Kernig’s sign ( an indicator of subarachnoid hemorrhage or meningitis), severe headache, vomiting, photophobia, chills

Meningococcemia: acute fever with chills, malaise, prostration, Waterhouse-Friderichsen syndrome (WFS)- is a group of symptoms resulting from the failure of the adrenal glands to function normally as a result of bleeding into the gland, Disseminated intravascular coagulation (DIC)

Other Syndromes: Pneumonia, arthritis, urethritis, respiratory tract infection, Waterhouse- Fredericksen syndrome, Meningococcal disease is favored by deficiency of the terminal complement components (C5-C9).

Epidemiology

The human is the only reservoir of the N. meningitidis. 5-10% of adults are asymptomatic carriers. Modes of infection are direct contact or respiratory droplets (nasopharyngeal carriers )from the nose and throat of infected people. The prevalence of meningitis is highest in the meningitis belt of Africa (frequent epidemics occurred there). In 1996, among 150000 cases 15000 deaths were reported. Epidemic usually occurs in overcrowded areas. Inhalation of contaminated droplets adherence of organism to nasopharyngeal mucosa. Local invasion and spread from the nasopharynx to meninges through the bloodstream (directly along the perineural sheath of the olfactory nerve, cribriform plate to subarachnoid space). In meninges, organisms are internalized into phagocytic cells. They replicate and migrate to subepithelial spaces and the incubation period is 3-4 days. Meningitis is more common in children below the age of 5 years and in males. Serogroup A, B, and C are responsible for outbreaks.

Laboratory diagnosis of Meningococcus

They are frequently isolated from samples such as blood and CSF. Other specimens petechial lesions, nasopharyngeal swab -especially to detect carrier may also be used. Examination of CSF: CSF should be turbid in this infection. The collected CSF is divided into 3 tubes: -Tube no 1 for chemical analysis( biochemistry) for glucose and protein estimation, tube 2 for microbiological tests ( microbiology) for Gram stain, latex agglutination test, and culture- sensitivity while tubing 3 records overall appearance -cell count ( WBCs in hematology). Microscopy: Gram-stained smear of CSF deposit commonly shows Gram-negative intracellular diplococci. White cell count increases to several thousand per cubic mm or µl with 90-99% polymorphonuclear cells (PMNs). Biochemical tests: Glucose is markedly diminished while CSF protein is markedly raised. CSF Culture: Inoculated into the blood or chocolate agar and incubated at 37ºC in 5-10% Carbon dioxide (CO₂) and high humidity. After 24 hours bacterial colonies appear and the organism is tested for biochemical and agglutination reactions. Now in details, follow as-

Useful methods for laboratory diagnosis of Meningococcus are-

Gram staining
Culture
Biochemical test
Latex agglutination test
Serotyping
Molecular test
Electrophoresis

Gram staining: The diagnosis is suggested by the finding of gram-negative bacteria bean-shaped capsular ( mark of evidence in Gram stain) diplococci as shown above image but the capsular mark is not recovered gram stain from culture.

Culture: The organism is cultured on blood agar or chocolate agar incubated at 37°C in a 5% CO₂ atmosphere. Colonies are 1-2 mm in diameter, convex, grey and transparent, and have no hemolysis as shown above picture.

Biochemical test: Following biochemical tests are important for Meningococcus identification-

Catalase test: Positive

Oxidase test : Determines the presence of cytochrome oxidase. It is Positive in N. meningitidis. Grow the isolate(s) to be tested for 18-24 hours on a blood agar plate at 35-37°C with 5% CO₂. Dispense a few drops of Kovac’s oxidase reagent. Tilt the plate and observe colonies for a color change to purple. Positive reactions will develop within 10 seconds in the form of a purple color. Another method-Use a platinum wire or wooden stick to remove a small portion of a bacterial colony (preferably not more than 24 hours old) from the agar surface and rub the sample on the filter paper or commercial disk. Observe inoculated area of paper or disk for a color change to deep blue or purple within 10 seconds as shown above image.

Fermentation test: Ferments glucose and maltose with acid production

Nitrate test: negative

Colistin resistant

Do not ferment lactose, sucrose and fructose

Gamma-glutamyl aminopeptidase test: positive

DNAase test: Positive

Latex agglutination test: It detects capsular polysaccharides in the CSF.

Serotyping: Serogroups and serotypes on the basis of the specificity of capsular polysaccharide antigens divided into 13 serogroups. These are A, B, C, D, X, Y, Z, W -135,29-E, H, I, K and L. Serogroups A, B, C, X, Y, W 135: most commonly associated with meningococcal disease Group A: epidemics Group C: localized outbreaks Group B: both epidemics and outbreaks.

Based on the outer membrane protein serogroups further divided into serotypes- About 20 serotypes have been identified.

Molecular test: For Detection of Meningococcal DNA. Polymerase Chain Reaction (PCR) is very useful.

Electrophoresis: For detection of soluble polysaccharide antigen, counter-current immunoelectrophoresis (CIEP) is used.

Treatment and Prevention

Penicillin G or sulphonamides are the drugs of choice. Chloramphenicol or third-generation cephalosporins such as cefotaxime or ceftriaxone are recommended for patients who are allergic to penicillin. Meningococcal vaccine is available which contains the capsular polysaccharide.

Prophylaxis

Chemoprophylaxis: It is indicated for the close contacts of patients for eliminating the bacteria from the nasopharynx. Following antimicrobial agents are recommended-

Rifampicin
Minocycline and
Ciprofloxacin.

Immunoprophylaxis: A vaccine containing capsular polysaccharides of serotypes A and C: for infants below 2 years and a quadrivalent vaccine constituted by polysaccharides of serotypes A, C, Y, and W-135: for children and adults whereas conjugate vaccine: polysaccharide antigen is conjugated to Diptheria toxoid.

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